New Insights into the Lactic Acid Resistance Determinants of Listeria monocytogenes Based on Transposon Sequencing and Transcriptome Sequencing Analyses

ABSTRACT Listeria monocytogenes is a foodborne pathogen that can tolerate a variety of extreme environments. In particular, its acid resistance (AR) capability is considered one of the key factors threating food safety. Here, we employed a microbial functional genomic technology termed transposon sequencing (Tn-seq), leading to the identification of two genes involved in cell wall peptidoglycan biosynthesis (murF) and phosphate transport (lmo2248) that play key roles in lactic acid resistance (LAR) of L. monocytogenes. Deletion of lmo2248 significantly impaired the ability of LAR in L. monocytogenes, demonstrating the accuracy of the Tn-seq results. Transcriptome analysis revealed that 31.7% of the L. monocytogenes genes on the genome were differentially expressed under lactic acid (LA) treatment, in which genes involved in phosphate transport were influenced most significantly. These findings shed light on the LAR mechanisms of L. monocytogenes, which may contribute to the development of novel strategies against foodborne pathogens. IMPORTANCE Listeria monocytogenes is a Gram-positive foodborne pathogen with high lethality and strong stress resistance, and its strong acid tolerance leads to many foodborne illnesses occurring in low-pH foods. Lactic acid is a generally recognized as safe (GRAS) food additive approved for use by the FDA. However, the genetic determinants of lactic acid resistance in L. monocytogenes have not been fully identified. In this study, the lactic acid resistance determinants of L. monocytogenes were comprehensively identified by Tn-seq on a genome-wide scale. Two genes, murF (cell wall peptidoglycan biosynthesis) and lmo2248 (phosphate transport), were identified to play an important role in the lactic acid resistance. Moreover, genome-wide transcriptomic analysis showed that phosphotransferase system (PTS)-related genes play a key role at the transcriptional level. These findings contribute to a better understanding of the lactic acid resistance mechanism of L. monocytogenes and may provide unique targets for the development of other novel antimicrobial agents.

This is a concise manuscript that studies the lactic acid tolerance response of the foodborne pathogen Listeria monocytogenes through transposon mutant library profiling (Tn-seq) and genome-wide gene expression analysis (RNA-seq). While technically correct, the manuscript may need to be improved further as described below: Major points: The introduction should be adapted to include more text on our current knowledge of lactic acid tolerance mechanism in L. monocytogenes and previous studies that have identified loci in the Listeria monocytogenes genome that contribute to this response, e.g. Begley  The pH of the medium with the different concentrations of lactic acid should be measured and reported in the manuscript. (L. 94 -101) L. 241. Significantly more detail should be provided on how the mutant and the complemented strain were generated. In addition, more detail could be provided to describe how the transposon mutant library was generated and validated (l. 263).
Sequence data needs to be uploaded to a data repository (e.g. the Short Read Archive) Minor points: While I had no problems understanding the manuscript, there are some minor grammatical or textual issues throughout which could benefit from corrections.
L. 58: all use of antibiotics will select for resistance, not just abuse.
L. 67-73: it is better to give a specific, relevant examples rather than adding 'etc' as that is not particularly informative.
L. 115 and L. 198 STRING analysis shows whether genes are interacting with each other, not necessarily whether they are related to each other as that suggests they are homologs, which I believe is not what the others mean to say. Have homologs of this gene been characterised in other Gram-positive bacteria (e.g. Bacillus subtilis, Lactobacillus or Lactococcus)?
L. 121 It is confusing that murF is apparently an essential gene, but the authors do identify a transposon mutant in that gene. How can this be explained?
L. 124 Complementary should be complemented L. 155. This is the first time the abbreviation DEGs is used and it should be explained here.
In the section on the RNA-seq analysis, it should be mentioned whether lmo2248 was significantly up-or down-regulated to connect these analyses with the Tn-seq data presented previously in the manuscript.

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Response to reviewers' comments
Dear Prof Luxin Wang, On behalf of my co-authors, we thank you very much for giving us an opportunity to revise our manuscript, we appreciate editor and reviewers very much for their positive and constructive comments and suggestions on our manuscript entitled "New insights into the lactic acid resistance determinants of Listeria monocytogenes based on Tn-seq and RNA-Seq analyses".
We have studied the comments of editor and reviewers carefully and have made revision which marked in red in the revised manuscript. We have tried our best to revise this manuscript according to the comments. Attached please find the revised version, which we would like to submit for your kind consideration. And responses to reviewers' comments are appended below.
We would like to express our great appreciation to you and reviewers for comments on our paper. Looking forward to hearing from you. List of Responses. Reviewer #3: This is a concise manuscript that studies the lactic acid tolerance response of the foodborne pathogen Listeria monocytogenes through transposon mutant library profiling (Tn-seq) and genome-wide gene expression analysis (RNA-seq). While technically correct, the manuscript may need to be improved further as described below. Reply: We thank the reviewer for his/her compliments on our manuscript. Below we will reply in a point-by-point fashion to the reviewer's comments and indicate the alterations we have made to the manuscript.